NADPH oxidases in vascular pathology

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NADPH Oxidases in Vascular Pathology

Anna Konior,¹Agata Schramm,¹Marta Czesnikiewicz-Guzik,^1,2and Tomasz J. Guzik^1,2

Abstract

Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of ‘‘kindling radicals,’’ which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall.

Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2.

Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions:

Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814.

Introduction

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zymes. Initial generation of ROS by NADPH oxidases triggers the release of ROS from other sources (109). NADPH oxidase homologues are differentially expressed in the vascular wall, including endothelial cells, smooth muscle cells (SMCs), fi- broblasts, and infiltrating immune cells (110). The expression profile of NADPH oxidases varies not only between different disease states, but also at various stages of the disease such as atherosclerosis. In general, it is accepted that under physio- logic conditions, vascular NADPH oxidases have a relatively low level of constitutive activity. However, enzyme activity can be increased both acutely and chronically in response to stimuli such as cytokines (38), growth factors (23), hyperlip- idemia, and high glucose (94), which disrupts vascular ho- meostasis and results in pathology.

While the role of vascular NADPH oxidases has been well described in pathology, their physiological functions

1Department of Internal Medicine, Jagiellonian University School of Medicine, Cracow, Poland.

2Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom.

Volume 20, Number 17, 2014 ª Mary Ann Liebert, Inc.

DOI: 10.1089/ars.2013.5607

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remain less clear. We have recently gained substantial in- sight into the contribution of individual NADPH oxidase homologues in the maintenance of normal vascular func- tion. In particular, the role of Nox4 in the regulation of endothelial function was clearly defined (166). This review focuses on the role of vascular NADPH oxidases in physi- ological and pathological processes in the vasculature, with particular emphasis on recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, and tu- mor angiogenesis. Finally, we briefly discuss the possibil- ities of pharmacological regulation of vascular NADPH oxidases and inhibitors being developed, in both the labo- ratory and clinical wards.

Localization, Structure, and Basic Functions of Major Nox Isoforms in Vasculature

Vascular Nox isoforms have six transmembrane domains, including alpha helices with cytosolic N- and C-termini, which participate in electron transfer, leading to the reduction of molecular oxygen to superoxide anion. Electron flow and thus ROS production is tightly controlled by the interactions of Nox subunits with other proteins, subunit phosphoryla- tion, or elevation of intracellular calcium (15). There are seven isoforms of NADPH oxidases expressed in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2. Four (Nox1, Nox2, Nox4, and Nox5) are most commonly expressed in vascular cells, while other homologues have not been found or are expressed at very low levels; thus, their role has not been established so far.

Nox2

Initially termed gp91phox, it has been cloned and identified as a phagocytic respiratory burst oxidase, critical for the initial nonspecific host defense. In addition to phagocytes, it is the most widely expressed vascular NADPH oxidase isoform. It is expressed in vascular smooth muscle cells (VSMCs), ad- ventitial fibroblasts, endothelial cells, and perivascular adi- pocytes (92, 149, 188). This NADPH oxidase homologue has been characterized in detail and consists of the following subunits: gp91phox (glycoprotein-91 kDa phagocytic- oxidase, newly termed Nox2), p22phox, p47phox, p67phox, p40phox, and the GTPase Rac1. The gp91phox and p22phox subunits are membrane bound and together form cytochrome b558, located in cytoplasmic vesicles and the plasma mem- brane (20). The structure of this oxidase in vascular cells is similar to that found in phagocytes, although it may have additional or different regulatory subunits in selected condi- tions. In particular, Nox organizer protein 1 (NoxO1) and Nox activator protein 1 (NoxA1), initially discovered as Nox1 regulators (in place of p47phox and p67phox, respectively), may also have modest activating properties toward Nox2 (100). In endothelial cells, Nox2-derived ROS are important for p38 MAP-kinase-mediated proliferation and vascular en- dothelial growth factor (VEGF)-induced migration (187).

Nox2 that is expressed in endothelial cells is involved in the regulation of numerous functions of the endothelial cell. For example, Nox2 activation affects NO bioavailability, and modulates expression of adhesion molecules during inflam- mation and angiogenesis. These will be further discussed next.

Nox1

Nox1 (Mox1, NOH1) has been identified as the first ho- mologue of Nox2 (7). It shares a 60% amino-acid sequence identity with Nox2. Similar to its phagocytic homolog, Nox1 contains six transmembrane domains and conserved motifs corresponding to binding sites of heme, flavin, and NADPH.

Nox1 is expressed in endothelial, smooth muscle, and ad- ventitial cells of the vasculature. Most of the studies using recombinant Nox1 protein show localization in cell mem- branes, particularly in the plasma membrane (85). Using im- munofluorescence microscopy, endogenous Nox1 protein displayed surface distribution along the cellular margins where it co-localized with caveolin-1 (33, 87). Other studies showed endogenous or overexpressed Nox1 protein localized to the nucleus, cytosol (28), endoplasmic reticulum (ER) (2), and mitochondria (40). Nox1 activity requires p22phox, NoxO1 (or possibly p47phox in some cases), and NoxA1, and the small GTPase Rac. Nox1-dependent ROS generation has been shown to play a pivotal role in cell signaling, cell growth, angiogenesis, and cell motility (6). Interestingly, ROS- generated via Nox1 have been reported to contribute to a growing number of diseases involving vasculature, including atherosclerosis, hypertension, neurological disorders, in- flammation, and cancer (35, 142), which will be further dis- cussed next. However, while highly expressed in animal models of vascular disease, Nox1 appears to be only ex- pressed at low levels in human peripheral or coronary vessels (79, 80).

Nox4

This Nox isoform is a 67 kDa protein sharing a 39% amino- acid sequence identity with Nox2. It was initially detected in the kidney; therefore, it was termed Renox or kidney oxidase (KOX) (62). However, it was soon identified in vascular walls, particularly VSMCs, fibroblasts (15), and endothelial cells (64, 65). Studies of human coronary arteries have shown Nox4 to be expressed predominantly in the media (170). It has been suggested to be involved in stress signal transduction in the kidney (67) and SMCs (151). Nox4 mediates transforming growth factor b (TGF-b)-induced differentiation (173), insulin signaling (130), oxygen sensing (115), cardiac differentiation (116), and transcriptional regulation (107). Nox4 is predomi- nantly localized at the ER (142, 151) and in the nucleus (40, 166). In addition, co-localization of Nox4 with the cytoskele- ton and focal adhesions has been demonstrated (36, 87, 128).

Enzymatic activity of Nox4 mainly depends on the expression level of Nox4 and p22phox and likely does not need any further activation (131). Currently, it is unclear which type of ROS is predominantly generated by Nox4. Unlike Nox1 or Nox2 that primarily produce O2-

c, Nox4 has been shown to produce hydrogen peroxide (H2O2) (43, 85). Production of H2O2, rather than O2-

c, by Nox4 is possible due to a highly conserved histidine residue in the E-loop of Nox4 that pro- motes rapid dismutation of O2-

c before it leaves the enzyme (177). This aspect of Nox4 biology needs to be further clarified.

Physiological roles of Nox4 differ depending on cell type and stimulus. Nox4 might have an antagonistic function to Nox1 and Nox2 (166). Nox4 is implicated in both pro- and anti- apoptotic pathways. It is needed for 7-ketocholesterol- in- duced apoptosis in SMCs (151), while silencing of the protein induces apoptosis in pancreatic cancer cells (136). Nox4 is

VASCULAR NADPH OXIDASES 2795

important for the differentiation of cardiac cells from mouse embryonic stem cells (116) and myofibroblasts from cardiac fibroblasts (37). Differentiated VSMC require Nox4 to main- tain expression of differentiation markers, smooth muscle major histocompatibility complex (MHC), alpha -actin, and calponin (36). Recent studies raise important questions re- garding the major physiologic and pathologic roles of Nox4.

While Nox4 contributes to oxidative stress, compelling evi- dence from Nox4^{- / -} mice indicates that endogenous Nox4 protects the vasculature during ischemic or inflammatory stress (166). Interestingly, we have recently found that Nox4 expression is decreased in human abdominal aortic aneurysm (AAA) in spite of largely increased oxidative stress in the walls of AAAs (74). Moreover, Nox4 immunoreactivity has been demonstrated in the nucleus and nucleolus of VSMCs by confocal microscopy (87). A recent study has identified a no- vel nuclear-localized Nox4 splice variant with a size of 28-kDa, Nox4D, which lacks putative transmembrane do- mains (3). The possible functional role of nuclear Nox4 has been previously described (87). Interestingly, Nox4D over- expression results in increased NADPH-dependent ROS production and causes increased phosphorylation of extra- cellular signal-regulated kinase1/2 and the nuclear tran- scription factor Elk-1 (3). Thus, Nox4D may have important pathophysiologic effects through the modulation of nuclear signaling and DNA damage; however, its role in vascular biology should be further elucidated.

Nox5

Nox5 is expressed in primates but does not naturally occur in rodents. It has been found in leukocytes, in the testis (during development), and in lymphatic tissue. Expression of lesser extent has been reported in ovaries, pancreas, and placenta (8, 32). Recent studies have also identified the pres- ence of Nox5 in vasculature, namely in endothelial cells and VSMCs, where it is localized in the ER and the plasma membrane (16). Nox5 has been reported to produce both su-

peroxide and H2O2; Nox5 is activated by Ca^{2 +} and does not appear to require other subunits, although it may associate with p22phox. The basic transmembrane structure of Nox5 is similar to that described for other Nox isoforms, but what distinguishes Nox5 is a unique N-terminus which encodes four calcium-binding EF hands (Fig. 1) (8). We have identified a calcium-dependent Nox5 protein and mRNA in human coronary vasculature, in both endothelium and vascular me- dia, which was functionally linked to calcium-dependent NADPH-oxidase activity (75).

Diverse Mechanisms of Activation and Regulation of Vascular NADPH Oxidases

Despite similarities in core structures, vascular Nox ho- mologues have different mechanisms of activation. Activation as well as its consequences may be different in the endothe- lium (Fig. 2) or VSMCs (Fig. 3) or other cells such as peri- vascular adventitial fibroblasts or adipocytes Nox1 and 2 require association with cytosolic components (p47phox, p67phox or NoxO1 and NoxA1), Nox4 is constitutively active, and Nox5 is activated by an elevation in intracellular Ca^{2 +}. The activation mechanism of Nox2 is most clearly defined and similar to that described in phagocytes. On activation, p47phox is phosphorylated and translocates to the mem- brane, through the formation of a complex with p67phox and p40phox. Phosphorylation of p47phox induces a conforma- tional change in a tandem SRC Homology 3 (SH3) domain that enables binding to a proline-rich region in the cytosolic C-terminus of the transmembrane subunit p22phox. In- dependently, the GTP-binding protein Rac also moves to the membrane and activation occurs (15). In contrast to Nox2, the Nox4-based oxidase appears to be constitutively active and does not require p47phox, p67phox, or Rac.

Sustained activation of vascular NADPH oxidases occurs in response to numerous agonists. Vascular NADPH oxidases are responsive to several growth (platelet-derived growth factor [PDGF], epidermal growth factor [EGF], and TGF-b),

FIG. 1. Nox5 expression and function in human ath- erosclerosis. Based on Guzik et al. (75).

cytokines (e.g., tumor necrosis factor, interleukin-1 [IL-1], and platelet aggregation factor), mechanical forces (cyclic stretch, laminar, and oscillatory shear stress), metabolic factors (hy- perglycemia, hyperinsulinemia, free fatty acids, and ad- vanced glycation end products), and G protein–coupled receptor agonists (serotonin, thrombin, bradykinin, en- dothelin, and angiotensin II [Ang II]) (71, 152, 154). In addi- tion, c-Src, p21Ras, protein kinase C (PKC), phospholipase D, and phospholipase A2(PLA2) have been demonstrated to play key roles in signaling involved in vascular NADPH oxidase activation (1, 12, 39).

Ang II is one of the key agonists stimulating Nox1 and Nox2 oxidase subunit expression as well as oxidase activation in vascular cells and neutrophils (148, 156, 187). However, its effects on Nox4 and Nox5 are much less pronounced and perhaps indirect, which will be further discussed next (37, 111, 139). Ang II-induced production of ROS by VSMCs is initially PKC-dependent (early phase), whereas the prolonged phase ( > 30 min) is dependent on Rac, Src, and phosphatidylinositol 3-kinase (167). Ang II increases mRNA levels not only of Nox proteins but also of p22phox and p67phox (55, 148). Ang II- dependent or H2O2-induced activation of Nox4 in mesangial cells or cardiac fibroblasts has been reported to be dependent on PLA2and release of arachidonic acid (37, 86).

Cytokines have also been shown to regulate vascular NADPH oxidases, which links inflammation with oxidative stress. In particular, tumor necrosis factor-a (TNF-a) stimu- lates NADPH oxidase Nox1, Nox2, and Nox4 expression and activation in a variety of vascular cells (4, 11, 138). Other stimuli that induce vascular NADPH oxidases include ER stress (151), shear stress (91), or an elevation of intracellular Ca^{2 +}, which could act as an upstream signal to activate Nox during cellular stress. The Nox5 isoform contains multiple EF- hand Ca^{2 +}-binding domains in the N-terminal region, al-

lowing its activation through calcium sensing (8). Calcium binding induces a conformational change in the N terminus of Nox5, resulting in the exposure of a hydrophobic motif, which leads to a direct interaction between the regulatory N- terminus and the catalytic C-terminus. This intra-molecular interaction is likely to be responsible for Ca^{2 +}-induced Nox5 activation (9), as it enables electron flow to the heme moieties and, consequently, drives superoxide production.

Redox regulation of vascular NADPH oxidase activation provides both negative feedback and feed-forward regulation mechanisms. For example, Rac1 protein turnover is strongly dependent on the redox status of the cell, creating a negative feedback mechanism (106). At the same time, a feed-forward mechanism exists (118), by which exogenous exposure of SMC or fibroblasts to H2O2-activated NADPH oxidase stim- ulates them to produce endogenous O2-

c, thereby amplifying the vascular injury process. The self-limiting mechanism could predominate during physiologic conditions, and be involved in maintaining low output of the nonphagocyte NADPH oxidase, whereas the feed-forward mechanism may have a role in NADPH oxidase-dependent oxidative stress in a variety of diseases, including atherosclerosis and inflammation.

NADPH Oxidases in Vascular Pathobiology

The complexity of vascular NADPH oxidase systems makes it difficult to unequivocally define the role of individ- ual Nox oxidases in vascular physiology and pathology.

Several genetically modified mouse strains with either re- moved or overexpressed Nox proteins have been used to in- vestigate the roles of NADPH oxidases in the vasculature (Table 1). Such studies have clearly linked Nox1, Nox2, and Nox4 to the pathogenesis of vascular diseases, while other FIG. 2. Selected interactions be-

tween NADPH oxidases in the endothelium. While certain func- tions were demonstrated for selected homologues, high redundancy of functions is expected. To see this il- lustration in color, the reader is re- ferred to the web version of this article at www.liebertpub.com/ars

VASCULAR NADPH OXIDASES 2797

Nox isoforms showed little to no vascular expression or in- volvement. Obviously, this approach is not optimal for studies involving Nox5, which would require a more complex humanized model approach. Numerous studies in animal models and in humans have also characterized the role of Nox proteins in the most common vascular disease states such as atherosclerosis, hypertension, and diabetes, which will be discussed next.

Atherosclerosis

A number of studies have investigated the role of indi- vidual NADPH oxidases in atherosclerosis in animal models.

Nox2 levels were increased in the aortas of apolipoprotein E (ApoE)^{- / -} atherosclerotic mice, while Nox4 levels were not significantly different between ApoE^{- / -}and wild type (97).

However, studies using deletion of the Nox2 gene in ApoE^{- / -} mice have not been fully consistent. Nox2/ApoE double knockout mice, which had been fed a high fat diet, were found to have no obvious protection against atherosclerosis when examining aortic sinus sections, in spite of decreased vascular superoxide formation (102). Similar surprising results were reported in global p47phox knockout mice crossed with

ApoE^{- / -} mice (90). Endothelial-targeted Nox2 over- expression in ApoE^{- / -} mice was sufficient to increase vas- cular superoxide production, endothelial cell activation, and subsequently increased macrophage recruitment. This initial increase in macrophage recruitment did not alter the pro- gression of atherosclerosis (50). In contrast to these studies, Barry-Lane et al. found that total aortic lesion area (from arch to bifurcation) was reduced in p47phox knockout mice crossed with ApoE^{- / -} mice, suggesting that Nox1 and/or Nox2 may be involved in atherogenesis (10). Such dis- crepancies in the findings regarding the vascular conse- quences of p47phox^{- / -} could be due to several reasons. An example is the inclusion of assessments of different areas from aortic lesions (sinus vs. whole aorta). Another reason for the differences may also be related to the removal of p47phox, which causes immunodeficiency. These mice are prone to sub- clinical infections that are characterized by splenomegaly and inflammation which could counteract the anti-atherosclerotic effect of the loss of NADPH oxidase. While studies in animal models are conflicting, a genetic hereditary deficiency of NADPH oxidases, particularly Nox2 (gp91phox), occurs in humans with chronic granulomatous disease (CGD). In hu- mans, the genetic deficiency of Nox2 is associated with FIG. 3. Diversity of functional consequences of the activation of NADPH oxidases in vascular smooth muscle cells and perivascular tissues. To see this illustration in color, the reader is referred to the web version of this article at www .liebertpub.com/ars

enhanced endothelium-dependent flow-mediated vaso- relaxation, and decreased markers of vascular aging and oxi- dative stress (192). Importantly, diminished atherosclerosis burden has been described in these patients, which empha- sizes the possible role of Nox2 oxidase in atherosclerosis (191).

Although promising, these observations should be inter- preted carefully, as CGD is a serious disease leading to im- portant changes in the immune system, which could affect vasculature in a number of additional ways.

Nox4 may also participate in atherogenesis, although much less is known in this regard. It has been shown, however, that Nox4 regulates VSMC migration and differentiation, which is critical for neo-intima formation (128). In human atheroscle- rosis, Nox4 expression is increased in intimal lesions of cor- onary arteries (170). Similarly, Nox1 overexpression in the media results in increased neo-intima formation on vascular injury (114).

Numerous studies have looked at vascular NADPH oxi- dases in human atherosclerosis. Our group as well as others have characterized NADPH oxidases in human coronary ar- teries with evident atherosclerotic plaques (75, 79, 170), as well as in peripheral arteries and veins without plaques but characterized by systemic endothelial dysfunction (73, 74, 80–

82). The NADPH oxidase is predominantly Nox2-based in veins, whereas an Nox4-based oxidase appears proportio- nately more important in human mammary or radial arteries (80). In veins, the predominance of Nox2 expression may also suggest the major contribution of endothelium and adventitia to total vascular ROS production, as these contain Nox2- based oxidases (14), potentially contributing to endothelial dysfunction (160, 170, 175). However, the cellular expression of individual oxidases has not been clearly established. While

initial studies in human vessels suggest that Nox2 is not usually present in medial VSMCs, it has been demonstrated that human microvascular SMCs can express Nox2 in re- sponse to Ang II (182). Meanwhile, Nox4 expression is in- creased by oscillatory versus pulsatile flow, which could be particularly relevant to the development of atherosclerotic plaques in the regions of turbulent flow (91). This clearly il- lustrates that Nox isoform expression in human vascular cells is regulated in a complex manner that depends on the cell type predominating in different vessels and on the nature of pathophysiologic stimuli.

Still, it is vital to emphasize that overall, enzymatic sources of O2-

c in coronary arteries are similar to those of peripheral arteries (18, 78). In summary, Nox2, Nox4, Nox5, p22phox, and, to a lesser extent, Nox1 are expressed in human pe- ripheral and coronary vessels, which may contribute to ath- erosclerosis (80, 170). Nox1 expression directly alters cell proliferation (174), and treatment of VSMC with Ang II or PDGF up-regulates Nox1, while down-regulating Nox4 (111).

Vascular injury increases expression of Nox1, Nox2, and p22phox, while Nox4 increases later (175), coinciding with a reduction in the rate of VSMC proliferation. Taken together, these findings suggest that while Nox1 and Nox2 are involved in acute response to injury or to Ang II stimulation, Nox4 is involved in maintaining the quiescent VSMC phenotype (175). Thus, a high Nox2/Nox4 ratio in veins could partially account for the susceptibility to intimal hyperplasia, re- modeling, and accelerated atherosclerosis in vein grafts (197);

whereas a lower Nox2/Nox4 ratio in mammary artery grafts could convey less susceptibility to atherosclerosis. Functional consequences of increased NADPH oxidase activity also in- clude the effects on lipid oxidation, which is a critical step in Table1. Selected Mouse Models Used to Study the Importance of Vascular

NADPH Oxidases and Their Functional Effects

Disease Mouse model Effect on disease References

Atherosclerosis Double knockout Nox2^{- / -}/ApoE^{- / -}

No change in lesion area in the aortic sinus Kirk et al. (102) Double knockout

p47^{- / -}/ApoE^{- / -}

No change in lesion area in the aortic sinus; Hsich et al. (90) Decreased total aortic lesion area Barry-Lane et al. (10) Nox2 endothelium-specific

overexpression in ApoE^{- / -}

Increased, vascular inflammation, and oxidation but not atherosclerosis.

Douglas et al. (50) Nox1 VSMC overexpression Increased neointima on wire injury Lee et al. (114) Hypertension Nox1^{- / -} Decreased BP and O2-

levels Matsuno et al. (132), Gavazzi et al. (60)

Nox1 VSMC overexpression Increased BP Dikalova et al. (44)

Nox2^{- / -} No change in BP Touyz et al. (182)

Decreased BP Jung et al. (98),

Wang et al. (193)

Nox4^{- / -} No change in BP Kleinschnitz et al. (103)

p47^{- / -} Decreased BP and O2-

c levels Landmesser et al. (108), Lavigne et al. (113) Pulmonary

hypertension

Nox4^{- / -} No change in pulmonary blood pressure

but prevention of poststroke neurodegeneration

Kleinschnitz et al. (103)

Cardiac failure Nox1^{- / -}; Nox2^{- / -} Effect on infarct size in myocardial ischemia/reperfusion; Decreased

hypertrophy; Increased cardiac hypertrophy

Braunersrenther V et al.

(23a)

Bendall et al. (17) Byrne et al. (25)

Nox4^{- / -} Decreased cardiac hypertrophy and failure Kuroda et al. (107)

ApoE, apolipoprotein E; NADPH, nicotinamide adenine dinucleotide phosphate; VSMC, vascular smooth muscle cell.

VASCULAR NADPH OXIDASES 2799

the initiation of atherosclerotic plaque development. For in- stance, Nox4 expression elevates in relation to oscillatory flow and coincides with increased oxidative stress and low-density lipoprotein (LDL) oxidation (91), while NADPH oxidase activity is correlated with oxidized-LDL in carotid plaques.

Despite the differences in the expression levels between individual Nox homologues, the expression of both p22phox and Nox4 mRNA is strikingly correlated in human arteries and veins (80). This indicates that risk factors for athero- sclerosis may be critical for the systemic regulation of NADPH oxidase expression (80). Indeed, activity and ex- pression of major NADPH oxidase components is correlated to the number of major risk factors for atherosclerosis in humans (82).

Finally, Nox5 is an important source of ROS in athero- sclerosis (75). Nox5 mRNA and Nox5 protein are dramatically increased in human coronary arteries obtained from pa- tients with coronary artery disease. These correlate with the Ca^{2 +}-dependent NADPH oxidase activity in arteries. Nox5 was expressed in the endothelium of early-stage lesions and in VSMCs in the intima of advanced coronary lesions (Fig. 1) (75).

In summary, differential generation of ROS by functionally distinct NADPH oxidase isoforms expressed in different vascular cell types in atherosclerosis may be used as a thera- peutic advantage. For example, Nox2-based production of ROS predominates in the endothelium and adventitia, whereas Nox1 and/or Nox4 could be much more important in VSMC (158), as their expression and activity differs de- pending on the stage of the disease. Thus, one could postulate, that if we were able to specifically inhibit these individual oxidases, we would be able to affect distinct stages in vascular disease processes.

Hypertension

Vascular NADPH oxidases are probably characterized in most detail in relation to hypertension. This was one of the first pathologies in which NADPH oxidases were clearly im- plicated (156). Ang II plays an important role in the devel- opment of hypertension and is one of the most important inducers of increased NADPH oxidase-dependent superoxide production in VSMCs (48) and throughout the vascular wall (141). The effects of Ang II on NADPH oxidases are mediated primarily via the angiotensin receptor type 1 (AT1) receptor (79). NADPH oxidases also participate in Ang II-stimulated intracellular H2O2production, which mediates vascular hy- pertrophy (205). Ang II increases the expression of several NADPH oxidase homologues (Nox1, Nox2, Nox4, and p22phox) (156), all of which may to some extent participate in the pathogenesis of hypertension and associated vascular dysfunction. Nox1 is increased in the aortas of aged sponta- neously hypertensive rats (SHRs) in parallel with increased NADPH oxidase activity. While Nox2 was also up-regulated, Nox4 protein levels were unchanged or even decreased in the aortas of SHRs (198). Overexpression of Nox1 in mouse VSMCs caused a marked increase in systolic blood pressure and hypertrophy in response to Ang II (44). In contrast, deletion of Nox1 in mice results in a blunted hypertensive response to Ang II and protects from development of endo- thelial dysfunction, oxidative stress, vascular hypertrophy, and aortic dissection (60, 61, 132). Overexpression of p22phox

in VSMCs leads not only to up-regulation of Nox1-based oxidase in the vessel wall, but also to increased hypertensive responses to Ang II (112, 195). At the same time, silencing of p22phox in rats prevents a slow pressor response to Ang II (137). Similarly, p47phox knockout mice, which have deficient Nox1 (and Nox2) activation, exhibit reduced hypertension and preserved endothelial function after chronic Ang II treatment (108, 109). VSMCs from p47phox-deficient mice do not produce superoxide (113). Moreover, local overexpression of Nox1 in VSMCs has profound effects on other cell types in the vessel wall. For example, this can lead to the oxidation of tetrahydrobiopterin (BH4) in endothelial cells and endo- thelial nitric oxide synthase (eNOS) uncoupling, resulting in a decrease of NO bioavailability and causing endothelial dys- function (45). In contrast, Nox2^{- / -} mice were reported to have only modestly altered (193) or even unaltered (183) blood pressure responses to Ang II, suggesting that this iso- form may be less important in blood pressure regulation, but still showing effects on vascular hypertrophy (193) and en- dothelial dysfunction during experimental hypertension (98). In addition, Nox2 was critical for cardiac hypertrophy in Ang II-dependent hypertension (17), but not in a pressure- overload-dependent model, where hypertrophy might be related to other homologues such as Nox4 (25). While gene- specific deletion or overexpression approaches have proved useful in defining the role of Nox1and Nox2 in the vascular system, the functional importance of Nox4, while undeniable, has been raising the most controversy and discussion.

Endothelium-targeted Nox4 overexpression causes enhanced vasodilation and reduced basal blood pressure, suggesting a protective role for Nox4 that has been linked to increased H2O2production by Nox4 (157). In the absence of pathogenic stimuli, Nox4 knockout mice do not have an obvious phe- notype and are normotensive (176). However, more recent data have convincingly shown that in vascular pathologies, Nox4 may serve as a protective oxidase (22, 166). In the con- ditions associated with stress induced by ischemia or by Ang II, loss of Nox4 resulted in reduction of eNOS expression, NO production, and heme oxygenase-1 (HO-1) expression. These changes were associated with apoptosis and inflammatory activation in Nox4^{- / -}mice (166). These effects were partially related to Nrf-2 induction. Consequently, there is now clear evidence that endogenous Nox4, in contrast to Nox1 and Nox2, may protect the vasculature during ischemic or hy- pertensive stress (166). However, this may not be the case in the brain, where Nox4 has been shown to contribute to oxi- dative stress related to stroke and other pathologic conditions (103). Hence, further studies are needed to determine whether Nox4 is always protective or whether it can become dys- functional.

Moreover, while the role of Nox1 in the pathogenesis of hypertension appears to be relatively well documented, chronic models of high renin transgenic overexpression do not show significant effects of Nox1 on blood pressure in this model of hypertension (204).

Diabetes

Diabetes is associated with a variety of metabolic abnor- malities, such as insulin resistance and hyperglycemia.

However, cardiovascular complications are the major cause of mortality in diabetic patients. ROS generated during

hyperglycemia are implicated in the development of endo- thelial dysfunction and progression of diabetic vascular complications. Endothelial dysfunction characterized by in- creased NADPH oxidase-dependent ROS generation has been demonstrated in animal models of diabetes (88), and in pa- tients with diabetes (82). In aortas from the streptozotocin (STZ)-induced diabetic ApoE^{- / -} mice, levels of Nox2 and Nox4 are increased (47). Similarly, in db/db mice, Nox1 or Nox4 are up-regulated, which is associated with increased ROS production and inflammation, indicating a potential role of Nox1 and Nox4 in diabetic macrovascular disease (119).

Another recent study has elegantly addressed the role of Nox1 in diabetic vasculopathy using the specific Nox inhibitor GKT137831, as well as Nox1^{- / -}mice on an ApoE^{- / -} back- ground in which diabetes has been induced (69). Deletion of Nox1, but not Nox4, had a profound anti-atherosclerotic effect correlating with reduced ROS formation, diminished che- mokine expression, reduced vascular adhesion of leukocytes, reduced macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers (69). Similarly, treatment of diabetic ApoE-deficient mice with GKT137831 attenuated atherosclerosis development (69).

Hyperglycemia itself can induce vascular NADPH oxi- dases. Incubation of human endothelial cells with red blood cells isolated from patients with type 1 diabetes, but not from normal volunteers, activated endothelial NADPH oxidase and increased endothelial ROS generation, which was medi- ated by advanced glycation end products on the surface of red blood cells from diabetic patients (194). Hyperglycemia increases NADPH oxidase expression, levels of oxidative stress markers, and apoptosis (155) of human endothelial cells in relation to increased NADPH oxidase subunit expression (for example, p22phox and p47phox) (46). Similarly, in ar- teries and veins, superoxide anion production is strongly in- creased in diabetic patients, most prominently in the endothelium (78). Superoxide anion produced in large quantities by NADPH oxidases reduce NO bioactivity by direct scavenging.

While NADPH oxidases play a key role, uncoupled eNOS has also been shown to be a particularly important source of ROS in diabetic blood vessels (88). Importantly, superoxide derived from vascular NADPH oxidases leads to eNOS un- coupling through oxidation of BH4 (88). Ang II is also in- volved in the activation of vascular NADPH oxidases in diabetes. AT1 receptor-mediated NADPH oxidase activation appears to contribute to vascular insulin resistance, endo- thelial dysfunction, apoptosis, and inflammation (196). While vascular Nox4 does not appear to have a direct role in diabetic vasculopathy (69), it may have indirect effects through the regulation of adipogenesis (preadipocyte differentiation in response to insulin) in metabolic syndrome (165).

Consequently, vascular NADPH oxidases have emerged as potentially important targets contributing to the pathogenesis of long-term cardiovascular complications of diabetes. Of the numerous pathologies and vascular disease states, diabetic vasculopathy may prove be the most interesting area for the future application of NADPH oxidase inhibitors.

In summary, genetically manipulated mouse models as well as studies in human vasculature have been very useful in defining the functional importance of individual NADPH oxidases. Further studies characterizing individual knockout and overexpression of Nox isoforms, specifically in endothe-

lial cells, VSMCs, and fibroblasts will undoubtedly provide a better understanding of the complex mechanisms involved in NADPH oxidase activation and regulation. Studies of Nox5 expression in various models are now warranted to further understand its role in the vasculature using these valuable molecular tools.

Vascular NADPH Oxidases in Pulmonary Hypertension Recently, significant progress has been made in our un- derstanding of the role of vascular NADPH oxidases in pul- monary hypertension. This is crucial, considering the clinical consequences of pulmonary hypertension and a lack of suc- cessful therapies. The major pathologic process in pulmonary hypertension is chronic hypoxia-induced vascular remodel- ing. It is characterized by malignant SMC hypertrophy and proliferation within the pulmonary vessel media (123). This leads to a decrease in vascular luminal area, increased vas- cular resistance, and thus development of pulmonary hy- pertension and increased right ventricular pressure. A role for Nox2-based NADPH oxidase in this process has been sug- gested, as all of these phenotypes are attenuated in Nox2^{- / -} mice (123). This is consistent with the role of this oxidase in VSMC hypertrophy, particularly pulmonary artery smooth muscle cells (PASMCs) (93, 173).

However, the role of NADPH oxidases in pulmonary hy- pertension appears to be more complex than simply stimu- lating VSMC hypertrophy and proliferation. They may be involved at very early stages of disease pathogenesis. ROS derived from vascular Nox isoforms, in particular Nox2 and Nox4, are involved in long-term responses of pulmonary vasculature to hypoxia (52, 135). Thus, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Inter- estingly, Nox4 has been reported to be the predominant ho- molog in human airways and in PASMCs (49). PASMCs are particularly sensitive to oxygen availability and are respon- sible for acute hypoxic vasoconstriction and the development of pulmonary hypertension in response to chronic hypoxia (72). Nox4 expression level has been increased in mice ex- posed to chronic hypoxia and in isolated PASMCs exposed to hypoxia. Diebold et al. have described a putative hypoxia responsive element in the human Nox4 promoter and dem- onstrated that this sequence is indispensable for the binding of hypoxia-inducible factor-1a (HIF-1a) (42). In fact, RNA inter- ference against HIF-1a suppressed the hypoxia-induced ex- pression of Nox4 (42). In patients with idiopathic pulmonary arterial hypertension, the level of Nox4 expression has been up-regulated in the lung. Treatment of PASMCs with an anti- Nox4 siRNA significantly reduced their proliferation.

Interestingly, it appears that Nox2 and Nox4 play coordi- nated roles in the development of hypoxia-induced pulmo- nary hypertension. Theoretically, endothelial ROS generation by Nox2 may stimulate Nox4 up-regulation in the vessel media, which would be important for hypoxia-dependent PASMC proliferation (135). Hypoxia also increases the level of TGF-b (97), which has been shown to induce Nox4 expression.

Inflammatory processes can also contribute to the induction of Nox4 in pulmonary hypertension. In particular, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB) is involved in the hypoxia-induced expression of Nox4 in PASMC, in that hypoxia increases the binding of NF-jB p65 to the putative NF-jB binding site adjacent to the putative

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Table2.ApproachestoInhibitVascularNADPHOxidases InhibitorBasicmechanismsofactionExamplesofvasculareffects ApocyninInhibitionofbindingp47phox subunittomembranecomplex(172).Unspecific scavengingofROSreportedincellswithoutMPO(86)Dose-dependentdecreaseofP-selectin,VCAM-1,plateletadhesion, monocyteaccumulationinLDL-R-/- (124) AttenuationofROSandhypertensioninrats(13) PreventionofNADPHoxidaseexpressionandactivationin fructose-fedanimals(168)andstreptozotocindiabetes(147) DPIUnspecificinhibitionofflavincontainingoxidases.Consideringitsverybroadspectrumofmetabolicactionsand toxicity,itisimpossibletouseitasagoodvascularNox inhibitorineithercellularorwholeorganismsystems(145,179)FormationofcovalentadductsInhibitionofNF-jBInhibitionof cholinesteraseactivityandinternalCa2+ pump gp91ds-tatInhibitionoftheinteractionofgp91phox andp47phox Potentininhibitingsuperoxidegenerationinthevascularsystem(160) TatpeptideoftheHIVvirus,allowsuptakeintothecellBlockingAngII-inducedO2- mouseaortaandattenuationofAng II-inducedhypertension(159) LackofactivityinpotentialoraladministrationlimitsitsutilityAmeliorationofendothelialdysfunctionbyreducingvascular superoxideandperoxynitriteintheDahlsalt-sensitive hypertension,butnosignificanteffectonbloodpressurein thismodel(207) VAS2870TriazolopyrimidineinhibitingROSproduction,initiallythoughttobeNox specific.RecentreportssuggestthatitmaynotinhibitNox-dependent ROSproduction(59)

InhibitionofROSinaortasofagedSHRs(199).Inhibitionof PDGF-stimulatedNADPHoxidaseactivityinratprimary VSMCs(180),andoxidizedLDL-mediatedROSformation inHUVECs(171) EffectsonNADPHoxidaseshavebeenrecentlyquestioned(59) S17834Syntheticpolyphenol,6,8-diallyl5,7-dihydroxy2-(2-allyl3-hydroxy 4methoxyphenyl)1-Hbenzo(b)pyran-4-one,Mechanism ofinhibition—unclear

Regulatorofadhesionmoleculeexpressionfortreatingchronic venousinsufficiency(189) ReducedTNF-a-stimulatedVCAM,ICAM-1andE-selectin expression,andreducedaorticatherosclerosisby60% inApoE-/- (27) AEBSFInterferencewiththeinteractionofp47phox and/orp67phox with cytochromeb559probablybychemicalmodificationofcyt(41)PreventsactivationoftheO2-generatingNADPHoxidase inbothintactstimulatedmacrophagesandincell-freesystems LackofspecificityinstudiesofvascularNADPHoxidases(199) CelastrolInterferencewithinteractionsbetweenthetandemSH3domainof p47phox andNOXO1andtheproline-richregionofp22phox extractedfrom Tripterygiumwilfordii,nonspecific

InhibitionofROSproductionbyNoxisoforms(Nox1and2vs. Nox4and5)(95).Inmousemicrovascularendothelialcells, CelastrolhasbeenreportedtodecreaseROSgenerationby Nox1inhibition(200) (Continued

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Table2.(Continued) InhibitorBasicmechanismsofactionExamplesofvasculareffects GKT137831InhibitionofNox1-andNox4-derivedROSproductionReductionofPPARcexpressionandH202generationaswellas cellproliferationinhumanpulmonaryarteryendothelialand smoothmusclecellsinpulmonaryhypertension(70) Preventionofhyperglycemia-inducedendothelialcelloxidativestress PreventionofatherosclerosisindiabeticApoE-/- (69) DualNox4-/Nox1-mediatedeffectsshouldbecarefullyobserved ML171PhenothiazineNox1inhibitorthatneedsfurtherspecificationPotentialuseinNox1inhibitionincoloncancerandvascular dysfunction(63) 17DMAGhsp90inhibitorDecreasedexpressionofNox1andNoxO1andreductionof NADPH-oxidaseactivityinVSMCsandmonocytes(129) DGLACyclooxygenasesubstratemetabolizedinmacrophagestoprostaglandinOralsupplementationdecreasesaorticlevelsofp22phoxand gp91phoxmRNAinApoE-/- mice(178) Tyrphostin AG490OralJak2inhibitor,DiminishesactivityandexpressionofaorticNox1,Nox2,andNox4 mRNAandproteininApoE-deficientmice(51) XJP-1,IsolatedfrombananapeelAttenuationofp22phoxandp47phoxexpressioninHUVECs(54) ModulationofthePI3K/Akt/eNOSpathway EXP3179InhibitionofPotentialinhibitionofNADPHoxidaseinhypertension(53) LosartanmetaboliteInhibitionofproteinkinaseC-dependentp47phox subunit phosphorylation 17DMAG,17-dimethylaminoethylamino-17-demethoxygeldanamycin;AEBSF,4-(2-aminoethyl)-benzenesulfonylfluoride;AngII,angiotensinII;DGLA,dihomo-gamma-linolenicacid;DPI, diphenyleneiodonium;eNOS,endothelialnitricoxidesynthase;HUVECs,humanumbilicalveinendothelialcells;ICAM-1,intercellularadhesionmolecule1;LDL,lowdensitylipoprotein;MPO, myeloperoxidase;NF-jB,nuclearfactorkappa-light-chain-enhancerofactivatedBcells;NoXO1,Noxorganizerprotein1;PDGF,platelet-derivedgrowthfactor;PPARc,peroxisomeproliferator- activatedreceptorgamma;ROS,reactiveoxygenspecies;S17834,6,8-diallyl5,7-dihydroxy2-(2-allyl3-hydroxy4-methoxyphenyl)1-Hbenzo(b)pyran-4-one;SH3,SRCHomology3;SHR,spontaneously hypertensiverat;TNF-a,tumornecrosisfactor-a;VCAM,vascularcelladhesionmolecule;XJP-1,7,8-Dihydroxy-3-methyl-isochromanone-4.

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